This invention is a further development in high-performance, high-pressure liquid chromatography, and relates generally to the solvent supply system for use in gradient elution liquid chromatography. In particular, this invention is concerned with the use of a single reciprocating pump for gradient elution liquid chromatography, and provides a technique for minimizing inaccuracies in the relative concentrations of the solvent components pumped from a plurality of reservoirs for passage through a chromatographic column.
Most prior art systems for gradient elution liquid chromatography used a separate pump for each of the components of the solvent mixture. The relative flow rates at which the pumps delivered their respective components to a mixing chamber determined the relative proportion of each component in the mixture. Typically, the pumping flow rates for the individual components were programmed to very temporally according to a desired schedule. In a two-component system for example, the concentration of the component supplied to the mixing chamber by one pump might be programmed to vary with time from 0% to 100% at a linear rate of 5% per minute. Pumps of various kinds were used, including reciprocating pumps or constant-displacement pumps of the syringe type.
Recently, single-pump systems have been developed for two-component gradient elution applications, with fast-acting valves serving to admit the two components to the pump chamber during the fill stroke according to a desired concentration ratio. Thus, in a two-component system, the duration of the fill stroke was shared between two proportioning valves according to the desired concentration ratio of the two components in the outflow from the pump. If a 20% concentration of one component in the outflow solvent mixture were desired, the corresponding valve admitting that component into the pump chamber would be opened for 20% of the duration of the fill stroke, while the other proportioning valve would be opened for the remaining 80% of the fill stroke duration. This sequencing of the opening and closing of the proportioning valves was repeated during successive fill strokes. The outflow from the pump was typically passed into a mixing chamber, wherein the mixture would homogenize.
Single-pump systems for use in gradient elution chromatography are described in U.S. Pat. Nos. 3,985,019 and 3,985,021, which have been assigned to the assignee of the present patent application. The liquid chromatography system described in these patents, the disclosures of which are incorporated herein by reference, includes the following: a chromatographic column; first and second reservoirs, each reservoir containing a particular solvent component to be introduced into the solvent mixture that serves as the mobile phase passing through the chromatographic column; reciprocating pumping means for causing the mobile phase to flow through the chromatographic column; motor means for driving the pumping means through successive pumping cycles, each pumping cycle comprising a fill stroke and a pumping stroke; first and second solenoid-actuated proportioning valves positioned at the low-pressure end of the pumping means, whereby components from the first and second reservoirs, respectively, are admitted into the pumping means according to a desired ratio; gradient setting means, whereby a temporally varying ratio for the components entering the pumping means from the first and second reservoirs can be preselected for a desired time interval; and means responsive to the gradient setting means, whereby the first and second proportioning valves can be actuated in complementary fashion with respect to their opening and closing so that the first valve is open while the second valve is closed during one portion of the fill stroke, and the first valve is closed while the second valve is open during another portion of the same fill stroke. Thus, it was known to the prior art to divide the fill stroke of a reciprocating pump into a first portion during which a first solvent component is pumped from a first reservoir, and a second portion during which a second solvent component is pumped from a second reservoir, according to a preselected temporally varying ratio as determined by a gradient setting means.
With single-pump gradient elution systems known to the prior art, in order to maintain the required flow rate for the solvent mixture through the chromatographic column, the reciprocating piston of the pumping means was required to travel back and forth at a relatively high velocity. The time duration of the fill stroke was independent of the speed of the reciprocating piston during the pumping stroke, and was typically selected to be approximately 0.2 second.
For convenience hereinafter, the solvent in the first reservoir shall be referred to as "component A," and the solvent in the second reservoir shall be referred to as "component B." At a time during the gradient elution program when substantially pure component A is required, i.e., when the concentration level of component B in the mobile phase outflow from the pumping means is substantially 0%, the proportioning valve associated with the second reservoir (hereinafter referred to as valve B) must remain closed throughout the 0.2-second duration of the fill stroke. As the temporally varying gradient elution changes to require a 1% concentration of component B, valve B is required to be open for approximately 0.002-second during the fill stroke, while the other valve (hereinafter referred to as valve A) remains open for the remainder of the fill stroke. Such precise time resolution is required in high-precision liquid chromatography, but is difficult to provide with mechanical proportioning valves. Thus, single-pump gradient elution systems known to the prior art tend to exhibit relatively large errors in gradient concentrations near the 0% and 100% concentration levels because of the inability of mechanical proportioning valves to provide the required time resolution.
Another factor that adversely affected the performance of single-pump gradient elution systems known to the prior art was the presence of finite residual volumes of solvent liquid within the pumping means after completion of the pumping stroke. Such residual liquid was compressed during the pumping stroke; and as the piston began to withdraw from the pump chamber in preparation for the next fill stroke, this compressed liquid tended to spit back into the particular component reservoir for which the corresponding proportioning valve was open. This spit-back effect was attributable to a lower pressure in the component reservoir than in the pump chamber. Because of this spit-back effect, there was an uncertainty as to the exact instant in time at which the pump chamber would begin to fill from the reservoir. Since the entire fill stroke was on the order of only 0.2-second, and since the gradient elution program might require that the proportioning valve for the particular reservoir be open for only a very small portion (e.g., 1%) of the duration of the fill stroke, this uncertainty in the starting time for the admission of solvent from a particular reservoir into the pump chamber often resulted in very significant inaccuracies in the concentration ratio for the components of the solvent mixture, particularly near the 0% and 100% concentration levels.
Another factor that adversely affected the performance of single-pump gradient elution systems known to the prior art was the "spit-forward" phenomenon, which occurred when the pressure in a reservoir for which the corresponding proportioning valve was open was greater than the pressure in the pump chamber. In that situation, the observe of the spit-back phenomenon would occur, i.e., the component in the open reservoir would be sucked into the pump chamber. This suction of reservoir liquid into the pump chamber prior to the onset of the scheduled proportioning according to the gradient setting means often resulted in significant inaccuracies in the concentration ratio of the solvent components, particularly near the 0% and 100% concentration levels.